Diabetic patients exhibit elevated serum TNF levels. We show in vitro that ten days after the induction of diabetes in rats, TNF stimulates Na and reduces Ca transport in isolated DT cells. In vivo clearance studies demonstrate that diabetic rats excrete more TNF and Ca and less Na in urine. TNF excretion in urine is significantly increased after the induction of diabetes and precedes increases of urinary Ca and decreases of Na excretion. When rats are administered an inhibitor of TNF synthesis, urinary excretion of TNF and Ca is reduced and Na excretion approximates that of control rats. In cell culture studies, the changes in Na and Ca transport occur with TNF treatment of cells not high glucose conditions. When cells from diabetic rats are maintained in culture in the absence of TNF, transport of Na and Ca returns is normalized comparable to that of controls. The goal of this proposal is test if anti-TNF agents prevent or attenuate altered transport and identify changes in gene expression and signaling mechanisms of TNF receptors that regulate DT Na and Ca transport. In Aim 1, we treat diabetic rats with selective agents that block TNF binding to receptors and determine if altered transport can be attenuated or reversed. We analyze urinary excretion of Na, Ca, and proteins and isolate cells from rats to assess transport and structure in DT cells. In Aim 2, gene expression will be examined using array technology and solution hybridization to identify genes activated by TNF in DT cells from diabetic rats and compare the response cells to those of controls. In preliminary studies, inhibitors of transcription and translation block TNF-induced Na absorption. Changes in gene expression are likely due to activation of the transcription factor NF-kappaB that modulates expression of receptors, signaling intermediates, or Na channel subunits. In Aim 3 we identify signaling pathways initiated or altered by TNF that acutely regulate transport of Na and Ca. We show that TNF activates a MAPK pathway to increase Na entry through epithelial Na channels. In contrast, TNF reductions in Ca transport occur through a sphingosine pathway. Specific inhibitors of these signaling mechanisms reverse the effects of TNF on DT cells. We use treated and untreated rats and in vivo measurements combined with biochemical, molecular, and pharmacological in vitro approaches to quantitate protein and receptors, signaling intermediates, and ion transport. Complementary techniques are used to examine alterations in vivo and at the cellular level. Our studies provide insights into the earliest changes that occur in DT due to the cytokine TNF.